Identification and quantification of phenolic compounds in grapes by HPLC-PDA-ESI-MS on a semimicro separation scale

Reversed phase high performance liquid chromatography (RP-HPLC) on a semimicro separation scale was employed to develop a straightforward method for the simultaneous separation, identification, and quantification of phenolic compounds occurring in whole berries of Vitis vinifera, which comprise phenolic acids, flavonols, catechins, stilbenes, and anthocyanins. A C-18 narrow bore column of 150 x 2.0 mm I.D. and a semimicro photodiode array detector (PDA) cell of 2.5 microL, in conjunction with a mass spectrometry detector equipped with an electrospray ionization source (ESI-MS) to confirm peak identification, were employed. The C-18 narrow bore column was eluted by a multisegment gradient of increasing concentration of acetonitrile in water-formic acid solution that was optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention times and both UV-visible and mass spectra, acquired by a mass spectrometer (MS) equipped with an electrospray ionization (ESI) source, employed in conjunction with the PDA detector. Libraries comprising retention times, UV-visible, and mass spectra for major phenolic compounds expected in grape berries were made by subjecting solutions of each phenolic standard to the optimized RP-HPLC method. Quantification of individual compounds was performed by the external standard method using a six point regression graph of the UV-visible absorption data collected at the wavelength of maximum absorbance of each analyte determined by the PDA spectra. The RP-HPLC method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. The developed method was successfully applied to identify the phenolic compounds occurring in the whole berries of nine red and one white grape of different varieties of Vitis vinifera, comprising some autochthonous varieties of south Italy such as Aglianico, Malvasia Nera, Uva di Troia, Negroamaro, Primitivo, and Susumaniello. Large differences in the content of phenolic compounds was found in the investigated grape varieties. As expected, only glycosilated flavonols were quantified, and the total amount of these compounds was higher in the whole berries of red grapes than in the white Moscato, where the most abundant phenolic compound was quercetin 3-O-glucoside. In almost all samples, the most and least abundant anthocyanins were malvidin 3-O-glucoside and cyanidin 3-O-glucoside, respectively, with the exception of Uva di Troia where the least abundant anthocyanin was delphinidin 3-O-glucoside.     

<Emphasis Type="Italic">Urochloa ruziziensis</Emphasis> cover crop increases the cycling of soil inositol phosphates

Ruzigrass (Urochloa ruziziensis) is a cover crop that is commonly used in Brazil and exudes high concentrations of organic acids from its roots, and is therefore expected to mobilize soil organic P such as inositol phosphates. However, it is not known if this can occur only under P deficient conditions. Specifically, we aimed to test the hypothesis that the degradation of inositol phosphates is increased by growing ruzigrass at two different P levels. To investigate this, we studied soil organic P in a 9-year-old field experiment, with treatments consisting of ruzigrass or fallow during the soybean (Glycine max) off-season, with or without P addition. Organic P was extracted in NaOH-EDTA, followed by colorimetric quantification of organic P hydrolysable by phytase, and myo-inositol hexakisphosphate by hypobromite oxidation and HPLC separation. Ruzigrass dry matter yield increased by about 80% with P application. Ruzigrass reduced the concentration of phytase labile P and myo-inositol hexakisphosphate, but only in soil receiving P. A corresponding increase in unidentified inositol phosphates, presumably representing lower-order esters, was also observed after ruzigrass in soil with P application. We deduce that the degradation of inositol phosphates under ruzigrass with P application is due to greater ruzigrass productivity in the more fertile treatment, increasing the release of root exudates that solubilize inositol phosphates and promote their decomposition by phytase. We conclude that ruzigrass cover cropping can promote the cycling of recalcitrant soil organic P, but only when fertility is raised to a sufficient level to ensure a productive crop.     

Nitrate,arsenic, cadmium,and lead concentrations in leafy vegetables: expected average values for productive regions of Chile

High arsenic (As), cadmium (Cd), lead (Pb), and nitrate (NO₃^?) concentrations in soil pose a risk for the human population and compromise food safety. The goal of this study was to obtain preliminary approximations for the expected mean values of As, Cd, Pb, and N-NO₃ in three leafy vegetables (lettuce, spinach, and chard) grown in the central farming regions of Chile. Representative samples (n = 148) of these crops were collected from the Coquimbo, Valparaíso, and Metropolitana Regions. Water extraction and an ion-selective electrode were used to determine NO₃^? contents, while total As, Cd, and Pb contents were determined by atomic absorption spectroscopy. The recorded values were 23.8%, 59.2%, and 97.9% below detection limits for As, Cd, and Pb, respectively. Furthermore, As and Cd intake by leafy vegetables reached 0.26 and 2.30% of the daily intake levels estimated by Chilean authorities. The daily NO₃^? intake by leafy vegetables was 0.44 mg per kg of bodyweight per day (kgbw^?1day^?1), or 12% of the WHO-recommended intake. No analyses were performed for Pb due to highly left-censored data. While the recorded NO₃^?, As, and Cd concentrations in lettuce, spinach, and chard do not apparently pose a health risk, further detailed studies are suggested.